Rbx2 E3-Ligase is Crucial for Bone Formation

Objectives: Ubiquitination regulates cell proliferation and differentiation by targeted protein degradation. A cascade of three enzymes is required to ubiquitinate the target protein. Runx2 and Sp7 are essential proteins for osteoblast differentiation and bone formation. Altered cellular levels of Runx2 and Sp7 proteins lead to cleidocranial dysplasia and osteogenesis imperfecta. Ubiquitination regulates Runx2 and Sp7 protein turnover, but the role of RING-box E3 ligase remains unknown. The goal of this study is to investigate the requirements of the RING-box E3 ligase, Rbx2, in embryonic and postnatal skeletal development.
Methods: Molecular, cellular, and histologic approaches were used to determine the skeletal phenotype of littermates lacking the Rbx2 gene in osteoblasts.
Results: We initially evaluated the spatial expression of Rbx2 in the newborn hindlimbs by RNAScope analysis. The expression of Rbx2 was noted in the developing and mature osteoblasts. To delete the Rbx2 gene, we bred Rbx2^F/F mice with the 2.3kb Col1a1-Cre line. Osteoblasts restricted activity of the Col1a1-Cre was confirmed using Td-reporter mice. The wildtype (Rbx2^+/+,Col1+) and homozygous (Rbx2^F/F,Col1+) littermates were analyzed. The Rbx2^F/F,Col1+ mice were born alive and survived to adulthood. H&E analysis of newborn femurs revealed that the loss of Rbx2 in osteoblast increased trabecular bone. Von kossa/alcian blue double-staining demonstrated increased mineralization in the Rbx2^F/F,Col1+ mice. To evaluate postnatal skeletogenesis, μCT analysis was performed on 1-month-old littermates. Interestingly, we find an increase in cortical BV, no change in TV, and an increase in BV/TV ratio in the Rbx2^F/F,Col1+ littermates. The trabecular bone showed an increase in BV, a reduction in TV, and an increase in the BV/TV ratio. Additionally, trabecular number and spacing increased while thickness decreased. The gene expression, histology, µCT analysis, and ex-vivo cultures are in progress to further quantify impairments in the Rbx2-deficient osteoblasts.
Conclusions: The Rbx2 enzyme is critical during embryonic and postnatal skeletal development.